preparation and hplc applications of rigid macroporous organic polymer monoliths

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J. Sep. Sci. 2004, 27, 747–766 www.jss-journal.de i 2004WILEY-VCH Verlag GmbH&Co. KGaA,Weinheim

Frantisek Svec

Department of Chemistry,University of California, Berkeley,CA 94720-1460, USA

Preparation and HPLC applications of rigidmacroporous organic polymermonoliths

Rigid porous polymer monoliths are a new class of materials that emerged in theearly 1990s. These monolithic materials are typically prepared using a simple moldingprocess carried out within the confines of a closed mold. For example, polymerizationof a mixture comprising monomers, free-radical initiator, and porogenic solventaffords macroporous materials with large through-pores that enable applications in arapid flow-through mode. The versatility of the preparation technique is demonstratedby its use with hydrophobic, hydrophilic, ionizable, and zwitterionic monomers. Sever-al system variables can be used to control the porous properties of the monolith overa broad range and to mediate the hydrodynamic properties of the monolithic devices.A variety of methods such as direct copolymerization of functional monomers, chemi-cal modification of reactive groups, and grafting of pore surface with selected polymerchains is available for the control of surface chemistry. Since all the mobile phasemust flow through the monolith, the convection considerably accelerates mass trans-port within the molded material, and the monolithic devices perform well, even at veryhigh flow rates. The applications of polymeric monolithic materials are demonstratedmostly on the separations in the HPLC mode, although CEC, gas chromatography,enzyme immobilization, molecular recognition, advanced detection systems, andmicrofluidic devices are also mentioned.

Key Words: Polymeric monoliths; Preparation; Modification; Application; Stationary phase; Sep-aration; HPLC; CEC;

Received: January 20, 2004; revised: April 20, 2004; accepted: April 20, 2004

DOI 10.1002/jssc.200401721

1 Introduction

Monoliths are separation media in the format that can becompared to a single large “particle” that does not containinterparticular voids typical of packed beds. The firstattempts to make “single-piece” separation media dateback to the late 1960s and early 1970s. For example,highly swollen monolithic polymer gel was prepared by

free-radical polymerization of an aqueous solution of 2-hydroxyethyl methacrylate with 0.2% ethylene dimeth-acrylate (crosslinking monomer), inserted into a glasstube, and used for size-exclusion chromatography in 1967[1]. Unfortunately, the effectiveness of fractionation wasrather low. Another early approach involved open-porepolyurethane foams prepared in situ [2–5]. In contrast tothe hydrogel, the permeability of these monoliths wasexcellent. However, excessive swelling in some solventsand softness were deleterious characteristics that pre-vented their successful use in both liquid and gas chroma-tography. Macroporous discs [6–9] and compressed softpolyacrylamide gels [10] placed in a cartridge or columnrepresent other examples of monolithic materials. Theseelegant approaches have been described in detail in aseries of excellent review articles [11–16]. The early1990s saw the development of another category of rigidmacroporousmonoliths formed by a very simple “molding”process in which a mixture of monomers and solvent waspolymerized and immediately used within a closed tube orother container under carefully controlled conditions [17].Since porous inorganic materials are very popular sup-ports widely used in catalysis and chromatography [18],monoliths prepared from silica were developed almostsimultaneously with the organic polymers [19, 20].

Correspondence: Frantisek Svec, Department of Chemistry,University of California, Berkeley, CA 94720-1460, USA.Phone: +1 510 643 3168. Fax: +1 510 643 3079.E-mail: [email protected].

Abbreviations: HPLC, high performance liquid chromatography;CEC, capillary electrochromatography; PEEK, poly(ether-ether-ketone); AMPS, 2-acrylamido-2-methyl-1-propanesulfonic acid;GMA, glycidyl methacrylate; VAL, 2-vinyl-4,4-dimethylazlactone;ST styrene; BuMA, butyl methacrylate; NIPAAm, N-isopropyl-acrylamide; AIBN, 2,29-azobisisobutyronitrile; TEMPO, 2,2,6,6-tetramethyl-1-pyperidinyloxy; carboxy-TEMPO, 4-carboxy-2,2,6,6-tetramethyl-1-piperidinyloxy; carboxy-PROXYL, 3-car-boxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy; TEMED, N,N,N,N-tet-ramethylethylenediamine; DEAE, diethylaminoethyl; THF, tetra-hydrofuran; HIC, hydrophobic interaction chromatography;LCST, lower critical solution temperature; ESI MS, electrosprayionization mass spectrometry; IP-RP-HPLC, ion-pair reversed-phase high-performance liquid chromatography; ODS, octade-cylsilica; SEC, size-exclusion chromatography.

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Detailed accounts of these materials have been publishedrecently [16, 21–23] and the newest developments arepresented elsewhere in this issue.

2 Macroporous polymersMacroporous polymers emerged in the late 1950s as aresult of the search for polymeric matrices suitable for themanufacture of ion-exchange resins with better osmoticshock resistance and faster kinetics. The history of theseinventions has been reviewed a short time ago [24]. Incontrast to the polymers that require solvent swelling tobecome porous, macroporous polymers are characterizedby a permanent porous structure formed during their prep-aration that persists even in the dry state. Their internalstructure consists of numerous interconnected cavities(pores) of different sizes, and their structural rigidity issecured through extensive crosslinking. These polymersare typically produced as spherical beads by a suspensionpolymerization process [25–27]. To achieve the desiredporosity, the polymerization mixture should contain both acrosslinking monomer and an inert diluent, the poro-gen [28–31]. Solvating or non-solvating solvents for thepolymer that is formed, supercritical carbon dioxide, orsoluble non-crosslinked polymers or mixtures of suchpolymers and solvents have proven to be efficient poro-gens.

Macroporous polymers are finding numerous applicationsas both commodity and specialty materials. The formercategory includes ion-exchangers and adsorbents, sup-ports for solid phase synthesis, polymeric reagents, andcatalysts, while chromatographic packings fit well into thelatter [32]. Although the vast majority of current macropor-ous beads are based on styrene-divinylbenzene copoly-mers, other monomers including acrylates, methacry-lates, vinylpyridines, vinylpyrrolidone, and vinyl acetatehave also been utilized [32].

While the suspension polymerization that affords macro-porous polymers has already been analyzed in the litera-ture many times [27–31], little was known until recently onhow to prepare macroporous polymers by bulk polymeri-zation within amold [17, 33, 34].

2.1 Preparation of rigid polymermonoliths

The preparation of rigid macroporous organic polymersproduced by a facile “molding” process is simple andstraightforward. The mold, typically a tube, is sealed atone end, filled with a polymerization mixture, and thensealed at the other end. The polymerization is then trig-gered, often by heating in a bath at a temperature of 55–808C [17, 35, 36]. In addition to thermally initiated poly-merization, redox initiation has also been used [37].Another option, UV light initiation, can only be carried outin UV transparent molds such as glass tubes, fused silica

capillaries, and microfluidic chips [38–41]. The seals arethen removed, the tube is provided with fittings, attachedto a pump, and a solvent is pumped through the monolithto remove the porogens and any other soluble compoundsthat remained in the pores after the polymerization wascompleted. A broad variety of tube sizes and materials,such as stainless steel, poly(ether-ether-ketone) (PEEK),glass, plastic microchips, and fused silica capillaries havebeen used as molds for the preparation of monoliths [37,38, 40–50].

While the preparation of cylindrical monoliths with a homo-geneous porous structure in capillaries and tubes up to adiameter of about 10–25 mm is readily achieved in a sin-gle polymerization step, larger size monoliths are some-what more difficult to prepare. Dissipation of the heat ofpolymerization is frequently slow and the “exotherm” maybe sufficient to increase substantially the reaction tem-perature, significantly accelerate the polymerization, andcause a rapid decomposition of the initiator. If this processis not controlled, monoliths with unpredictable radial andaxial gradients of porosity are obtained [44]. However, theslow and gradual addition of the polymerization mixture tothe reaction vessel in which the polymerization reactionproceeds continuously minimizes the heat production andallows the preparation of very large diameter monolithswith homogeneous porous structures. Another elegantmethod that helps to solve the problem of heat dissipationhas been demonstrated recently [51]. Using analysis ofthe heat release during the polymerization, Podgornik atal. derived a mathematical model for the prediction of themaximum thickness of the monolith that can be preparedin a single step without affecting the radial homogeneity ofthe material. To obtain large cylindrical objects, theseauthors prepared a few annular monoliths with variouswell-defined outer and inner diameters that inserted oneinto another to form a monolith with the desired largevolume. These radial flow columns extend the monolithictechnology to the field of scaled-up preparative separa-tions [52].

Buchmeiser et al. presentedanatypical approach tomono-lithic columns [53–56]. They used ring-opening meta-thesis copolymerization of norborn-2-ene and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalenewithin borosilicate glass columns in the presence of poro-genic solvents such as toluene, methylene chloride,methanol, and 2-propanol to obtain functionalized mono-lithic materials. A ruthenium catalyst was used to preparemonolithic separation media with typical macroporousmorphology. By variation of the polymerization conditions,such as the ratio ofmonomers, the porogenic solvents, andthe temperature, the pore size could be varied within abroad range of 2–30 lm, affording materials with specificsurfaceareas in a rangeof 60–210 m2/g.


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2.2 Control of porous properties

Many applications of porous materials in areas such ascatalysis, adsorption, ion exchange, chromatography,and solid phase synthesis rely on the intimate contact witha surface that supports the active sites. In order to obtaina large surface area, a significant number of smaller poresshould be incorporated into the polymer. The most sub-stantial contribution to the overall surface area comesfrom micropores, with sizes smaller than 2 nm, followedby the mesopores ranging from 2 to 50 nm. Larger pores(macropores) contribute very little to the surface area.However, these pores are essential to allow liquid to flowthrough the material at a reasonably low pressure. Thispressure, in turn, depends on the overall porous proper-ties of the material [36]. Therefore, the pore size distribu-tion of the monolith must be adjusted properly to fit eachtype of application.

The pore size distribution of the molded monoliths is quitedifferent from those observed for “classical” macroporousbeads. An extensive study of the types of pores obtainedduring polymerization both in suspension and in anunstirred mold has revealed that, in contrast to commonwisdom, there are some important differences betweenthe suspension polymerization used for the preparation ofbeads and the bulk-like polymerization process utilized forthe preparation of molded monoliths [35]. An example ofpore size distribution curves and the internal morpholo-gies recorded for both beads and monolith are shown inFigure 1. The morphology of the monolith featuring indivi-dual microglobules and their irregular clusters is similar tothat found for beads [57]. However, the size of the clustersand the irregular voids between them is much larger in themonolith. In the case of polymerization in an unstirredmold the most important differences compared to suspen-sion are the lack of interfacial tension between the aque-

ous and organic phases, and the absence of dynamicforces that are typical of stirred dispersions. The morphol-ogy of the monoliths is closely related to their porous prop-erties, and is also a direct consequence of the quality ofthe porogenic solvent as well as the percentage of cross-linking monomer and the ratio between the monomer andporogen phases. The presence of synergistic effects ofthese reaction conditions was verified using multivariateanalysis [38].

The porosity and flow characteristics of macroporouspolymer monoliths intended for use as separation mediafor chromatography, flow-through reactors, catalysts, orsupports for solid phase chemistry have to be adjustedduring their preparation. Key variables such as tempera-ture, composition of the pore-forming solvent mixture, andcontent of crosslinking monomer allow the tuning of theaverage pore size within a broad range spanning at leasttwo orders of magnitude from tens to thousands of nano-meters [34, 36, 58]. The scanning electron micrographs ofFigure 2 represent examples of the porous structures ofpoly(glycidyl methacrylate-co-trimethylolpropane tri-methacrylate) monoliths prepared using various polymeri-zation conditions [38].

The choice of pore-forming solvent (porogen) is themostly used tool for the control of porous properties with-out changing the chemical composition of the final mono-lith. In general, larger pores are obtained in a poorer sol-vent due to an earlier onset of phase separation. Theporogenic solvent controls the porous properties of themonolith through the solvation of the polymer chains inthe reaction medium during the early stages of the poly-merization [34, 36].

A large number of solvents has already been used to cre-ate the desired macroporosity in rigid monoliths [34, 36,59]. Supercritical carbon dioxide is the newest contribu-


Figure 1. Morphology and differen-tial pore size distribution curves ofpoly(glycidyl methacrylate-co-ethyl-ene dimethacrylate) beads andmonolith prepared from identicalpolymerization mixtures (adaptedfrom [58]). Polymerization mixture:glycidyl methacrylate 24%, ethylenedimethacrylate 16%, porogenic sol-vent cyclohexanol 48%, dodecanol12%, AIBN 1% (with respect tomonomers), temperature 708C,time 12 h. The pore size distributionwas determined by mercury intru-sion porosimetry; micrographs wereobtained using scanning electronmicroscopy.

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tion to the broad family of porogenic solvents [60, 61]. Thistype of porogen is attractive since it is non-toxic, non-flam-mable, and inexpensive. In addition, the properties of this“solvent” can be tuned by varying the pressure. Once thepolymerization is completed, the porogen is simply evapo-rated with no need for washing and no residual solvents inthe monolith. Using ethylene dimethacrylate and trimeth-ylolpropane trimethacrylate as monomers, a broad rangeof materials with typical macroporous structures and poresizes in a range of 20–8,000 nmwere prepared.

2.3 Hydrodynamic properties

For practical reasons, the pressure needed to drive theliquid through any system should be as low as possible.Because all of the mobile phase must flow through themonoliths, the first concern is their permeability to liquids,which depends completely on the size of their pores. Theextremely high pressures required to achieve flow throughwould likely damage a monolith with pores only of the sizefound in typical macroporous beads. Obviously, lower flowresistance can be observed for materials that have a largenumber of broad channels. However, many applicationsalso require a large surface area in order to achieve a highloading capacity. This high surface area is generally acharacteristic of porous material that contains smallerpores. Therefore, a balance must be found between therequirements of low resistance to flow and high surfacearea, and an ideal monolith should contain both largepores for convection and a connected network of shorter

and smaller pores for high capacity. Figure 3 shows theback pressure per unit of flow rate (resistance to flow) as afunction of the flow rate. Typically, the pressure needed tosustain even a very modest flow rate is quite high for mate-rials that have a mean pore diameter of less than about


Figure 2. Scanning elec-tron micrographs of theinner part of the poly(glyci-dyl methacrylate-co-tri-methylolpropane trimeth-acrylate) monoliths pre-pared from polymerizationmixtures varying in percen-tage of isooctane intoluene (porogenic sol-vent) and porogenic sol-vent/monomers ratio. Poresize: 35 (A), 225 (B), 4,990(C), and 6,890 nm (D) [38].

Figure 3. Effect of flow velocity on back pressure in a poly-(styrene-co-divinylbenzene) 10068 mm monolithic columnwith varying pore sizes. Conditions: Mobile phase tetrahydro-furan; polymerization mixture: styrene 20%, divinylbenzene20%. Varying toluene and dodecanol contents and polymeri-zation temperature; mean pore size 180 (1), 270 (2), 7365(3), and 7090 nm [36].


300 nm, while high flow rates can be easily achieved atlow pressures with monoliths that have pores larger than1,000 nm.

2.4 Surface chemistry

Obviously, the monolithic material may serve its purposeonly if provided with the surface chemistry required for thedesired application. For example, hydrophobic moietiesare well suited for reversed phase chromatography, ioniz-able groups must be present for separation in ion-exchange mode and CEC, and chiral functionalities arethe prerequisite for enantioselective separations. Severalmethods can be used to prepare monolithic columns witha wide variety of chemistries.

2.4.1 Preparation from functional monomers

The number of monomers that may be used in the pre-paration of polymer monolith is much larger than thoseused for classical suspension polymerization becausethere is only one phase in the mold. Therefore, almost anymonomer, including water-soluble hydrophilic monomers,which are not suitable for standard polymerization inaqueous suspensions, may be used to form a monolith.This greatly increases the variety of surface chemistriesthat can be obtained. However, the polymerization condi-tions optimized for one system cannot be transferreddirectly to another without further experimentation, and

the use of newmonomer mixtures always requires re-opti-mization of polymerization conditions in order to achievesufficient permeability of the resulting monolith [62]. A fewexamples of monomers (1–9) and crosslinking agentssuch as divinylbenzene (10), N,N 9-methylenebisacryl-amide (11), ethylene dimethacrylate (12), and trimethylol-propane trimethacrylate (13) that have been used for thepreparation of rigid porous monoliths are shown in Fig-ure 4. The list of monomers includes a broad variety ofchemistries varying from very hydrophilic acrylamide 8and 2-acrylamido-2-methyl-1-propanesulfonic acid 6(AMPS) through reactive such as glycidyl methacrylate 5(2,3-epoxypropyl methacrylate; GMA), chloromethylstyr-ene 2, 2-vinyl-4,4-dimethylazlactone 7 (VAZ) and pro-tected functionalities (4-acetoxystyrene 3), to ratherhydrophobic styrene 1 (ST) and butyl methacrylate 4(BuMA), and even zwitterionic 9 (N,N-dimethyl-N-meth-acryloyloxyethyl-N-(3-sulfopropyl)ammonium betaine)[62–69].

2.4.2 Modification of reactivemonoliths

Chemical modification is another route that increases thenumber of available chemistries, allowing the preparationof monoliths with functionalities for which monomer pre-cursors are not readily available. These reactions areeasily performed using monoliths prepared from mono-mers containing reactive group such as 2 and 5. For


Figure 4. Examples of monomersused for the preparation of rigidporous polymer monolithic col-umns.

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example, Figure 5 shows the reaction of poly(chloro-methylstyrene-co-divinylbenzene) with ethylenediamineand then with c-gluconolactone, which completelychanges the surface polarity from hydrophobic to highlyhydrophilic [70]. Very popular is the reaction of glycidylmethacrylate monoliths with diethylamine, which leads toan anion-exchanger suitable for the separation of pro-teins [17].

The living character of the ring opening metathesis poly-merization described earlier in this review enables a sim-ple preparation of functionalized norbornene-basedmonoliths. Adding one more in situ derivatization step thatinvolves functional norborn-2-ene and 7-oxanorborn-2-ene monomers that react with the surface-bound initiator,the pores are provided with a number of typical functionalgroups such as carboxylic acid, tertiary amine, or evencyclodextrin [53, 54].

Although glycidyl methacrylate-based monoliths mayreact directly with high molecular weight ligands such asproteins, this reaction is slow and inefficient [71]. There-fore, the monoliths are often modified to provide for morereactive functionalities. For example, Figure 6 shows aseries of reactions leading to an aldehyde functionalityand subsequent immobilization of Protein A affording amonolithic column for affinity chromatography [72].

2.4.3 Grafting of pore surface

The preparation of functionalized monoliths by copolymer-ization of functional monovinyl and divinyl monomersrequires optimization of the polymerization conditions foreach new set of functional monomers and crosslinkers inorder to obtain monoliths with the desired properties.Since the functional monomer constitutes both the bulkand the active surface of the monolith, a substantial per-centage of the functional units remains buried within thehighly crosslinked polymer matrix and is inaccessible forthe desired interactions. A better utilization of a preciousfunctional monomer might involve its graft polymerizationwithin large pores of a “generic” monolith. Using the sim-ple modification processes described in the previous sec-tion, only a single functionality is obtained from the reac-tion of each functional site of the monolith. In contrast, theattachment of chains of reactive polymer to the reactivesite at the surface of the pores would provide multiplefunctionalities emanating from each individual surfacesite, and thus dramatically increase the surface groupdensity. Such materials, which possess higher bindingcapacities, are attractive for use in chromatography, ion-exchange, and adsorption. M�ller has demonstrated thatthe cerium(IV) initiated grafting of polymer chains onto theinternal surface of porous beads affords an excellent sep-aration medium for biopolymers [73]. We used a similarreaction to graft AMPS 6 onto the internal surface ofhydrolyzed poly(glycidyl methacrylate-co-ethylene di-methacrylate) monoliths [64].

Grafting can also provide the monolithic polymers withrather unexpected properties. For example, Peters usedthe two step grafting procedure summarized in Figure 7,which involves the vinylization of the pore surface by reac-tion of the GMA epoxide moiety with allyl amine, and asubsequent radical polymerization of N-isopropylacryl-amide initiated by AIBN within these pores. This reactionsequence leads to a composite that changes its separa-tion ability in response to external temperature [74].

In an alternative approach, Tripp attached a free radicalazo initiator – 4,49-azobis(4-cyanovaleric acid) – to ben-zyl chloride functionalities at the pore surface of the poly-


Figure 5. Reaction scheme of hydrophilization of poly(4-chloromethylstyrene-co-divinylbenzene) monolith via reac-tion with ethylenediamine and gluconolactone [70].

Figure 6. Reaction scheme of immobiliza-tion of Protein A via hydrolysis of poly(gly-cidyl methacrylate-co-ethylene dimeth-acrylate) monolith followed by periodateoxidation, reaction with the protein, andreduction of the aldimine linkage [72].


(chloromethylstyrene-co-divinylbenzene) monolith. Sub-sequently, the graft polymerization of 2-vinyl-4,4-dimeth-ylazlactone was initiated from the surface. In order toavoid an undesirable increase in flow resistance and toimprove the yield of grafting, divinylbenzene was added tothe polymerization mixture to form a layer of swellablereactive polymer gel within the pores. These monolithswere used for the separation of various amines from solu-tions in flow-throughmanner [75, 76].

Stable free radical mediated crosslinking polymerizationhas also been used for the preparation of macroporousmonoliths [77, 78]. For example, the latent TEMPO-capped free radicals had a great potential for the prepara-tion of a variety of monoliths with different chemistries andenhanced capacities using grafting, provided the polymer-ization conditions could be modified to obtain monolithswith suitable porous properties. However, this polymeriza-tion using TEMPO as the stable free radical led first only toproductswith a less permeable porous structure as a resultof the rather high reaction temperature of 1308C requiredto obtain good conversions. In contrast, the use of “low”temperature mediators such as 2,2,5-trimethyl-3-(1-phe-nylethoxy)-4-phenyl-3-azahexane [79] in the preparationof porous monoliths substantially simplifies the control ofporous properties. As a result, polymers with a pore size of50–1100 nm can be prepared [80]. Viklund et al. carriedout polymerizations in the presence of yet different stablefree radical 3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy (carboxy-PROXYL) or 4-carboxy-2,2,6,6-tetramethyl-piperidinyl-1-oxy (carboxy-TEMPO) as mediators and abinary porogenic solvent consisting of polyethylene glycoland 1-decanol to prepare highly porous poly(styrene-co-divinylbenzene) monoliths [78]. These polymerizationswere found tobe fast and led tohigher degreesofmonomerconversions in a shorter period of time compared to corre-sponding TEMPO mediated reactions. The use of media-tors containing carboxylic functionalities accelerated the

reaction kinetics. Modification of the composition of poro-genic solvent then improved the permeability of the pre-paredmonoliths, and enabled control of the porous proper-tiesof themonolithic polymersover awide range.

The desired grafting of the preformed monoliths was thenachieved by filling the pores with a monomer solution andheating to the temperature required to activate the dor-mant radicals. For example, a functionalization of poly-(styrene-divinylbenzene) monolith with chloromethylsty-rene and vinylpyridine to obtain material with up to3.6 mmol/g of functionalities has been demonstrated [80].

Recently, we used UV initiated reactions to graft chainscomprising a wide variety of monomers onto the pore sur-face of monoliths. For example, Rohr prepared porouspoly(butyl methacrylate-co-ethylene dimethacrylate)monoliths with controlled pore size in fused silica capil-laries. Modification of the pore surface chemistry of thesemonoliths was then achieved via benzophenone initiatedphotografting to introduce poly(AMPS) and poly(VAL)chains [40]. In the case of AMPS, the grafting is very rapidand can bemonitored through the electroosmotic flow thatis afforded by the newly introduced ionized functionalities.The desired degree of loading with functional groups isobtained within only a few minutes. These monolithiccapillaries were successfully used for the isocratic separa-tion of peptides in electrochromatographic mode. Electronprobe microanalysis confirmed that grafting had occurredwith reasonably good radial homogeneity.

Using a similar procedure, Hilder grafted the pore surfaceof the monolith with N,N-dimethyl-N-methacryloyloxy-ethyl-N-(3-sulfopropyl)ammonium betaine 7 to obtain acapillary column for CEC separation of proteins [81]. Thisprocedure afforded monolithic columns that enabled rapidand reproducible separation of a variety of proteins withan extremely high efficiency of over 106 plates/m, albeitwith lower resolution and limited control of the EOF.

3 HPLC applicationsAlthough the history of monolithic polymers is relativelyshort, a number of applications has already beenexplored. These applications cover a rather broad rangefrom heterogenized catalysts [66, 71, 82–87], detec-tion [88], solid-phase extraction [41, 89], polymer-sup-ported chemistry [90, 91], molecular imprinting [92–99],to a variety of separations including gas chromatogra-phy [100], HPLC, and CEC. Most of these applicationshave been summarized in a recently published mono-graph [16]. The extent of this review does not allowdescription of all of them in detail; the main focus will be onseparations in the HPLCmode. Several detailed accountsof monolithic stationary phases for separations in the CECmode have also been published recently [81, 101–106]


Figure 7. Reaction scheme of modification of poly(glycidylmethacrylate-co-ethylene dimethacrylate) monolith with allyl-amine followed by free radical grafting of poly(N-isopropyl-acrylamide) chains [74].

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3.1 Reversed-phase HPLC of small molecules

Our early experiments with rigid monolithic poly(styrene-co-divinylbenzene) columns attempted separation of aro-matic compounds. The results indicated that the interac-tions between the hydrophobic surface of the monolithicstationary phase and solutes such as alkylbenzenes didnot differ from those observed with beads under similarchromatographic conditions [62]. The increase in averageretention, which reflects the contribution of one methylenegroup to the overall retention of a particular alkylbenzenesolute, had a value of 1.42 which was close to that pub-lished in the literature for typical polystyrene-basedbeads [107]. However, the efficiency of the early mono-lithic polymer column was only about 13,000 plates/m forthe isocratic separation of three alkylbenzenes. This valuewas much lower than the efficiencies of typical columnspacked with small polymer beads.

Recent papers published independently by two groups inthe Czech Republic demonstrate significant improve-ments in the HPLC separations. They both used similarporogenic mixtures consisting of 1-propanol, 1,4-butane-diol, and water, which we developed earlier for the pre-paration of monolithic columns for CEC. Moravcov� et al.prepared monolithic columns in 0.32 mm ID fused-silicacapillaries via AIBN initiated polymerization of BuMA andEDMA [48]. They characterized the through pores and themesopores containing stagnant mobile phase by meansof two variables, equivalent permeability particle diameterand equivalent dispersion particle size, respectively, thathave been introduced by Tallarek [108]. An increase in thepercentage of propanol in the polymerization mixture ledto a decrease in both pore volume and pore size in themonolithic media, with accompanying improvements incolumn efficiency. Unfortunately, this increase in perform-ance was achieved at the expense of decreased columnpermeability. Figure 8 shows separations of benzenederivatives using three monolithic columns differing in por-ous properties and length and compares them to the sep-aration in a column packed with ODS phase. The authorsof this study argue that monolithic columns exhibitedretention behavior similar to that of the column packedalkyl silica for compounds with varying polarities charac-terized by interaction indices; however, their methyleneselectivities were different. Higher concentrations of pro-panol in the polymerization mixture enhanced the lipophi-lic character of the monolithic stationary phases. Bothbest efficiencies and separation selectivities were foundfor monolithic columns prepared using porogenic mixturescontaining 62–64% propanol.

Holdsvendov� et al. used free radical polymerizationinitiated with redox system comprising ammonium perox-odisulfate and N,N,N,N-tetramethylethylenediamine toprepare monolithic poly(BuMA-EDMA) columns in

0.32 mm ID fused-silica capillaries [37]. For comparison,they also prepared similar columns via polymerizationinitiated thermally with AIBN. The reproducibility of the“redox” columns prepared at ambient temperature for theseparation of alkylbenzenes was good with RSDs of 1.1–3.6% for retention factor and 2.5–7.9% for HETP. Themost efficient column afforded a HETP value of 29 lm forunretained uracil which equals an efficiency of 35,000plates/m. Figure 9 demonstrates that the column effi-ciency and selectivity do not depend on the type of initia-tion. In contrast, the column prepared using the thermallyinitiated process exhibits a higher retention. The authorsalso claim that the preparation of monoliths using ammo-nium peroxodisulfate is easier to perform since it is carriedout at ambient temperature.

Despite recent improvements, the efficiency of the poly-methacrylate-based monolithic columns in the HPLC sep-arations of small molecules remains rather low comparedto efficiencies of about 105 plates/m that were observed


Figure 8. Isocratic separation of benzene derivatives usingthree monolithic columns varying in porous properties andcolumn packed with Biosphere C18 beads [48]. Mobilephase 70% aqueous ACN, flow rate 2.0–2.8 lL/min, UVdetection at 254 nm. Peaks: uracil (1), benzyl alcohol (2),benzaldehyde (3), benzene (4), toluene (5), ethylbenzene(6), propylbenzene (7), butylbenzene (8), amylbenzene (9).


for C18 modified silica-based monoliths reported byTanaka [20, 109]. Therefore further improvements of thepolymer-based monoliths are required. One of the targetsis the porous structure since that of silica and organicpolymer monoliths differs significantly. While the morphol-ogy of organic polymers consists of clusters of poorlyorganized microglobules with large pores located amongthem and with a low surface area, silica rods feature awell-ordered array of equally sized, about 1 lm largethrough-pores and skeletons. In addition, the skeletonsthemselves are mesoporous and provide the monolithwith a large surface area, a feature particularly valued inisocratic separations. This makes monolithic silica-basedcolumns suitable for rapid separations of small mole-cules [109, 110]. Therefore, a significant increase in theefficiency of the polymeric monolithic columns for the sep-arations of small molecules is likely to be achieved justthrough the optimization of their porous structure. Thissuggestions is also supported by the well-known fact thatthe quality of the bed structure has a decisive effect on thechromatographic properties of columns packed with parti-cles [18]. Similarly, the efficiency of electrophoretic sep-

arations has been found to reach its maximum for a speci-fic capillary diameter, while decreasing steeply in capil-laries with both larger and smaller sizes [111].

In contrast, very high column efficiencies can easily beachieved in the CEC mode with polymeric monoliths pre-pared from the same monomers as those used for theHPLC monolithic columns. For example, Yu demon-strated separation of benzene derivatives in the reversed-phase mode with an efficiency of over 200,000 plates/m [46] while L�mmerhofer achieved efficiencies of250,000 for the separations of enantiomers of modifiedamino acids in the ion-exchange mode [112]. This mightbe the result of much smaller pore sizes that can be usedfor monoliths in CEC columns.

3.2 Separation of synthetic oligomers

Early demonstrations of the separation of styrene oligo-mers by HPLC on ODS columns in a gradient of themobile phase follow the normal course well known forreversed-phase chromatography of small mole-


Figure 9. Isocratic separation of benzene derivatives using monolithic columns differing in porous properties [37]. Column A: hy-drophobicity index 3.6, total porosity of 0.79; Column D: hydrophobicity index 3.9, total porosity of 0.68. Mobile phase 65% aque-ous ACN, flow rate 2.0 lL/min, UV detection at 214 nm. Peaks: uracil (1), phenol (2), toluene (3), ethylbenzene (4), aniline (7).

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cules [113]. Their retention depends both on the composi-tion of the mobile phase and on the number of repeat unitsin the oligomer. Larger polystyrene oligomers, being morehydrophobic, are more retained. This means that shorteroligomers elute prior to longer ones, quite unlike size-exclusion chromatography for which larger moleculeselute first.

Petro was the first to demonstrate the separation of com-mercial styrene oligomers in a short monolithic column inthe gradient HPLC mode, and compared his process withthe separation achieved in the size-exclusion chromato-graphic (SEC) mode. The chromatograms were mirrorimages, and exhibited a number of peaks that could beassigned to the individual styrene oligomers [114]. Theresolution achieved with the monolithic column was verygood. It is well known that an increase in the resolution ofan SEC system can only be achieved with better columnpacking or a longer column. In contrast, gradient elutionmethods provide additional options for improving the sep-aration. If variables such as the range of mobile phasecomposition remain constant for a specific column and aspecific set of solutes, the average retention factor in gra-dient elution only depends on the gradient time and theflow rate [115]. Because the product of these variables isthe gradient volume, equal separations independent offlow rate and gradient steepness should be achievedwithin the same gradient volume. Figure 10 shows sep-arations of styrene oligomers on a monolithic poly(sty-rene-co-divinylbenzene) column obtained with gradienttimes of 200 and 20 min and flow rates of 1 and 10 mL/min, respectively. The gradient volume is 200 mL for bothseparations and, indeed, the separations are very similarwith only a small difference in resolution between the twochromatograms. This is also a clear demonstration of thepositive effect of convection on the separation of macro-molecular analytes.

In contrast to SEC, these results indicate that the addi-tional tools of flow rate and gradient time are available forthe optimization of separation in gradient elution chroma-tography [114]. Monolithic columns allow the use of veryhigh flow rates at reasonable back pressures, thus makingvery fast chromatographic runs possible. In addition, theyalso permit much higher sample loads than typical ODSpacked columns.

3.3 Precipitation-redissolution chromatographyof synthetic polymers

In this technique, originally developed for packed col-umns [116], the polymer solution is injected into a streamof the mobile phase in which the polymer is not soluble.Therefore, the macromolecules precipitate and form aseparate gel phase, which adsorbs onto the surface of theseparation medium and does not move along the column.The solvating power of the mobile phase is then increasedgradually until it reaches a point at which some of themacromolecules start to redissolve again and travel withthe stream. Since the medium contains pores smaller thanthe size of the polymer molecules, the mobile phase canpenetrate these small pores while the dissolvedmoleculesmove only with the stream through the larger channels. Asa result, the polymer solution moves forward faster thanthe solvent gradient, and therefore the polymer eventuallyprecipitates again. The newly formed precipitated gelphase will then redissolve only when the solvent strengthis once more sufficient. A multitude of such precipitation-redissolution steps is repeated until the macromoleculefinally leaves the column. The solubility of each polymermolecule in the mobile phase depends on both its molecu-lar weight and its composition. As a result, separation ofspecies differing in these properties is achieved.

Although higher molecular weight synthetic polymerssuch as polystyrene behave differently from small and


Figure 10. Effect of flow rate and gradient time on the separation of styrene oligomers in a molded poly(styrene-co-divinylben-zene) monolithic column (Reprinted with permission from ref. [114] Copyright 1996 American Chemical Society). Conditions: col-umn, 5068 mm ID. (a) mobile phase, linear gradient from 60 to 30% water in tetrahydrofuran within 200 min; flow rate, 1 mL/min.(b) mobile phase, linear gradient from 60 to 30% water in tetrahydrofuran within 20 min; flow rate, 10 mL/min; analyte, 15 mg/mLin tetrahydrofuran; injection volume, 20 lL; UV detection, 254 nm; peak numbers correspond to the number of styrene units inthe oligomer.


midsize molecules in reversed-phase chromatographicseparations, the general elution pattern from a monolithiccolumn remains unchanged, as the more soluble specieswith lower molecular weights elute prior to those withhigher molecular weights. For example, an excellent sep-aration of a mixture of eight narrow polystyrene standardswith molecular weights ranging from 519 to 2.956106 hasbeen achieved in a gradient of methanol in tetrahydro-furan even at a flow rate of 8 mL/min, despite the veryshort length (5 cm) of the column used. The separation iscompleted in a much shorter period of time at a higherflow rate. For example, 16 min are needed for the separa-tion at a flow rate of 2 mL/min, while only 4 min are suffi-cient for the same separation at 8 mL/min without compro-mising the quality of the separation. Once again, the gradi-ent volume required for the elution of a specific peakremains constant at both flow rates. In addition, the posi-tion of peaks in the chromatogram can be adjusted by asimple change of the gradient profile. Similar results wereobtained using mobile phases in which acetonitrile andwater were used as precipitants [114].

Generally, using higher flow rates and steeper gradientswith monolithic columns enables extremely fast separa-tions. This also applies to the precipitation-redissolutionchromatography of synthetic polymers. For example, theseparation of three polystyrene standards was carried outusing steep gradients and a flow rate of 20 mL/min [117].A very good separation was achieved at a gradient time of1 min, and three baseline resolved peaks were obtainedwithin 16 s. The speed of this method proved to be extre-mely valuable in the characterization of large libraries ofsynthetic polymers prepared using methods of combina-torial chemistry [16, 118].

Although successful, the separations described aboverequired high flow rate of 20 mL/min and consumed largevolumes of the mobile phase, thus limiting a broader useof this technique. Our recent study improved the applic-ability of monolithic columns for the rapid determination ofmolecular parameters of synthetic polymers using theprecipitation/redissolution technique since the separationcould then be completed at much lower flow rates. Thiswas achieved by combination of two factors: using col-umns with a smaller diameter and optimization of gradi-ents of the mobile phase. In addition to polystyrenes, sep-arations of poly(methyl methacrylates), poly(vinyl acet-ates), and polybutadienes have also been demonstratedand the results of these rapid separations compared withdata obtained using SEC [119]. For example, the separa-tion of nine polystyrene standards using a gradient thathas been optimized to obtain a linear calibration curvewas achieved in less than 2 min. This method also allowsthe rapid determination of molecular parameters of typicalpolymers with a broad molecular weight distribution,

affording results comparable with those obtained by muchslower SEC.

It is worth noting, that the commercial monolithic columnSwift RP-poly (Isco, Inc., Lincoln, NE) used in this studywas very stable. Over a period of about 7 months,approximately 3,000 chromatographic measurementswere carried out using a single 5064.6 mm ID poly(sty-rene-co-divinylbenzene) monolithic column. In each gra-dient run, one component of the mobile phase was a goodswelling agent for the material of the column while theother was a precipitant. Although the high level of cross-linking does not allow extensive swelling of the monolithicmaterial, even small volumetric changes of thematrix con-stitute a periodic stress for the column. However, thisrepeated stress had no effect on long-term column per-formance. During the course of this study the flow rate,one of the most critical variables, was changed quiteoften, routinely reaching values of up to 8 mL/min. Use ofsuch a high flow rate would not be feasible for a column ofthis size packed with HPLC grade particles due to the pro-hibitively high back pressure that would result. Great col-umn stability was also demonstrated by the use of severaldifferent solvents such as THF, dichloromethane, metha-nol, hexane, and water with repeated changes in gradientcomposition without any adverse effects on the separa-tions. An occasional low flow rate flushing with THF wasthe only “maintenance” carried out on the column. Duringthe entire period of study, no change in back pressure,flow, and separation characteristics was observed for themonolithic column. Figure 11 shows two HPLC separa-tions of a mixture of eight polystyrene standards recordedmore than two months and about 400 injections apart.Even after such a long time the very small difference thatcan be observed between these two runs lies within theexperimental error of chromatographic measurements[119].

3.4 Chromatography ofmidsize peptides

The rapid acceleration of research in the area of proteo-mics observed recently requires the development of newtechnologies. Peptide mapping is one of the methods thatappear ideally suited for both the identification of proteinsand the determination of posttranslational modifications.Typically, the protein of interest is digested by a proteolyticenzyme, most often trypsin, and the peptides in the result-ing mixture are identified using mass spectrometry. Thisprocess affords a peptide map that is unique for each pro-tein and allows identification through a search of existingdatabases. Obviously, even the most powerful currentmode of separation – reversed phase HPLC in gradientmode – does not have nearly the peak capacity requiredto separate proteome proteins or derived peptides to adegree sufficient for an accurate determination of compo-sition. Therefore, rapid and efficient multidimensional sep-


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aration methods in which the overall peak capacity is theproduct of peak capacities of the separation in eachdimensionmust be developed.

Despite its current limitations, HPLC remains a valuabletool for the separation of both proteins and peptides.Because of their higher molecular weights, the slower dif-fusional mass transport of the analytes within the pores oftypical porous beads in a packed column negativelyaffects the quality of the separation. In contrast, the sep-aration in a monolithic column is considerably faster,owing to the much better mass transport enhanced byconvection [120]. For example, the early isocratic separa-tion of the peptides with a molecular weight of about 1000such as bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) and (D-Phe7)-bradykinin, which differ only in their 7th

amino acid residue (L-proline and D-phenylalanine,respectively), was achieved within only 3 min using apoly(styrene-co-divinylbenzene) monolith and 50% aque-ous acetonitrile [62].

The ability to preparemonoliths within amold of any shapewas used by Moore et al. to prepare monolithic poly(sty-rene-co-divinylbenzene) stationary phase within pulledfused silica capillary needles for the reversed-phase sep-

aration and on-line electrospray mass spectrometrydetection of proteins and peptides [121]. As illustrated byFigure 12, these monolithic microcolumns separatedpeptides far better than capillaries packed with both com-mercial ODS and poly(styrene-co-divinylbenzene) beads.

Premstaller et al. prepared 6 cm long poly(styrene-co-divi-nylbenzene) monolithic columns in 0.2 mm ID capillaryformat using tetrahydrofuran/decanol as the porogenicsolvents [122]. These columns were tested for the rapidand highly efficient HPLC separation of protein digests fol-lowed by ESI MS detection, enabling protein identifica-tion [123]. As expected, they found that the loading capa-city of the column was a function of molecular mass andfor both small peptides and large proteins reached the0.4–0.9-pmol range. Using the UV detection and a 3 nLcapillary detection cell they could determine an octapep-tide even at 500 amol concentration. The applicability ofmonolithic column technology in proteomics was demon-strated by the mass fingerprinting of peptides formed bytryptic digestion of both bovine catalase and b-lactoglobu-lin, as well as human transferrin [123, 124].

Poly(styrene-co-divinylbenzene) monolithic columnswere also prepared via thermally initiated polymerizationin 75 and 125 lm ID PEEK, stainless steel, and fusedsilica capillaries using a mixture of propanol and forma-mide as the porogenic solvents [125]. In order to increasethe hydrophobicity of these columns, Friedel-Crafts reac-tion with chlorooctadecane in the presence of aluminumchloride as a catalyst was carried out in situ. Figure 13compares total ion chromatograms of tryptic digest usingboth original and alkylated poly(styrene-co-divinylben-zene) monolithic columns in gradient mode.

A significant improvement in the separation ability of themonolithic columns for peptides has been recentlydemonstrated by Ivanov et al. [49]. They explored the useof 20 lm ID poly(styrene-co-divinylbenzene) monolithiccapillary columns for the LC-ESI-MS analysis of trypticdigest peptide mixtures. The polymerization conditionsand mobile-phase composition were optimized for chro-matographic performance leading to efficiencies over100,000 plates/m for peptide separations. This group alsodemonstrated a high mass sensitivity in a range of about10 amol for peptides in the MS and MS/MS modes usingan ion trap mode. This represented a 20-fold improvementover 75 lm ID columns. Figure 14 shows an excellentgradient nano-LC-ESI-MS separation of a tryptic digest ofa mixture of ten proteins on the 10 cm long 20 lm IDmonolithic column with 10–40 fmol injected peptides. Forexample, the gradient separation of a proteolytic digest ofa tissue extract was carried out with a sample size equiva-lent to mere 1000 cells. The monolithic capillary columnafforded a wide linear dynamic range of almost 4 orders ofmagnitude, and good run-to-run and column-to-column


Figure 11. Rapid separation of a mixture of 8 polystyrenestandards using a 5064.6 mm ID monolithic poly(styrene-co-divinylbenzene) column Swift RP-poly (Isco, Inc.) and thecorresponding gradient profile monitored by the UV detector(Reprinted with permission from ref. [119]. Copyright 2000Wiley-VCH). Separation conditions: 1.25 min gradient ofTHF in methanol consisting of 0–35% THF in methanol in0.12 min, 35–50% in 0.38 min, 50–55% in 0.25 min, 55–59% in 0.25 min, and 59–60% in 0.25 min, overall sampleconcentration 16 mg/mL (2 mg/mL of each standard) in THF,ELSD detection. Molecular weights of polystyrene standards:3,000 (1), 7,000 (2), 12,900 (3), 20,650 (4), 50,400 (5),96,000 (6), 214,500 (7), and 980,000 (8). Dotted line showsthe same separation recorded more than two months andabout 400 injections apart



Figure 12. Base peak chromatograms for theLC/MS analyses of a cytochrome c Lys-Cdigest (0.7 pmol injected) using monolithicpoly(styrene-co-divinylbenzene) columnplaced in ESI needle (a), and needles packedwith C18 silica (Vydac 218 TP) (b), andPoros 10 R2 porous polymer beads (Re-printed with permission from ref. [121]. Copy-right 1998 American Chemical Society).

Figure 13. Performance of the unmodified (a) and octadecyl-ated (b) monolithic poly(styrene-co-divinylbenzene) columnsfor the separation of peptides from the tryptic digest of cyto-chrome c represented by the TIC chromatograms (Reprintedwith permission from ref. [125]. Copyright 1998 AmericanChemical Society). Column: 10060.125 mm ID, PEEK tub-ing. Sample: 0.7 pmol each of the peptides from the digest.Mobile phase A: 0.1% acetic acid and 0.01% heptafluoro-butyric acid in water; B: 0.1% acetic acid and 0.01% hepta-fluorobutyric acid in acetonitrile, Gradient of B in A: 0–10%in 1 min, 10–30% in 10 min, 30–60% in 15 min; flow rate0.3 lL/min; temperature 208C; voltage for ESI with thetapered fused-silica capillary +3.5 kV; MS detection in range380–1700m/z.

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reproducibility of separations in both isocratic and gradientelution modes.

3.5 Gradient elution of proteins

Gradient elution is a very popular method for the separa-tion of natural macromolecules because the retention ofdifferent components of a complex biological mixture mayvary considerably. In contrast to isocratic separations, theuse of a gradient of mobile phase accelerates the elution,allowing separation of the components to be achievedwithin a reasonable period of time. The mechanism of gra-dient elution is similar for many of the retentive HPLCmodes such as reversed-phase, ion exchange, and hydro-phobic interaction chromatography [115, 126]. Typically,the first step is the adsorption of the sample on the separa-tion medium close to the top of a column, followed by suc-cessive dissolution of individual components as the com-position of the mobile phase is changed. The nature of themobile phase is dictated by the separation mode used.For example, mixtures of water or a dilute buffer solutionand organic solvent such as acetonitrile are typically usedfor elutions from a highly hydrophobic separation mediumin reversed-phase chromatographic mode.

The monolithic media tolerate fast flow rates, thus easilyenabling high throughput separations. In fact, the excel-lent performance of our early monolithic columns wasdemonstrated just on reversed-phase separation of threeproteins using 10068 mm ID poly(styrene-co-divinylben-zene) monolith in the broad flow rate range of 5–25 mL/min while keeping the gradient volume constant. All indivi-

dual proteins were always baseline separated in sharpnarrow peaks. This, together with the low back pressureobserved even at very high flow rate, enabled acceleratedseparations to be achieved simply with an increase in theflow rate or by using an even steeper gradient [42, 127].Figure 15 shows the rapid separation of five proteins inless than 17 s using commercial monolithic column SwiftRP-pro (Isco, Inc.).

Poly(styrene-co-divinylbenzene) monolithic capillary col-umns developed by Huber’s and Henion’s groups wereconnected to a mass spectrometer and used for the sep-aration of proteins [124, 125]. Excellent separations re-quiring 10–15 min in a slow gradient mode were demon-


Figure 14. 3-D overlay of gradient nano-LC-ESI-MS chromatograms of a tryptic digest of a mixture of ten proteins (cytochrome c(horse), trypsinogen (bovine), myoglobin (horse), serum albumin (bovine), ovalbumin (chicken), b-casein (bovine), a-1-acid glyco-protein (human), b-lactoglobulin (bovine), a-lactoglobulin (human), and catalase (bovine)) on the monolithic poly(styrene-co-divi-nylbenzene) column (Reprinted with permission from ref.[49]. Copyright 1998 American Chemical Society). Mobile phase: (sol-vent A) 2% (v/v) acetonitrile, 0.1% (v/v) formic acid in water; (solvent B) 10% (v/v) water, 5% (v/v) 2-propanol, 0.1% (v/v) formicacid in acetonitrile. Gradient: 5% B, 0 min; 40% B, 25 min; 90% B, 26 min; gradient steepness parameter b = 0.28. Injection of10–40 fmol was made and data acquisition commenced 10 min after the start of the gradient.

Figure 15. Rapid reversed-phase separa-tion of proteins using a monolithic poly-(styrene-co-divinylbenzene)column SwiftRP-pro (Isco, Inc.) at a flow-rate of 10 mL/min (Reprinted with permission fromref.[127]. Copyright 1999 Elsevier). Col-umn: 5064.6 mm ID, mobile phase gradi-ent: 42 to 90% acetonitrile in water with0.15% trifluoroacetic acid in 0.35 min, UVdetection at 280 nm. Peaks: ribonu-clease (1), cytochrome c (2), bovineserum albumin (3), carbonic anhy-drase (4), chicken egg albumin (5).


strated with capillaries comprising both poly(styrene-co-divinylbenzene) and its alkylated counterpart monoliths.

We recently prepared monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns usingphotoinitiated polymerization within 200 lm ID capillariesand used them for the lHPLC separations of proteins inreversed-phase mode. The low resistance to flow, a gen-eral feature of monolithic columns, enabled rapid separa-tions at very high flow rates of up to 100 lL/min, represent-ing a flow velocity of 87 mm/s. Thus, a model protein mix-ture consisting of ribonuclease A, cytochrome c, myoglo-bin, and ovalbumin was baseline separated in less than40 s using a very simple single step gradient of the mobilephase (Figure 16). Interestingly, no effect of the pore sizewithin a range of 0.66–2.2 lm on the retention of proteinswasobserved for thesemonolithic columns [128].

Norbornene-based 5063 mm ID monolithic columnswere also used for the separation of model proteins inreversed phase chromatographymode [53]. Thesemono-liths easily tolerated high flow rates of up to 10 mL/minand a good separation of ten proteins in about 4 min con-firms a fast mass transfer, even at this high flow rate.

In contrast to reversed-phase chromatography, the sep-aration in ion exchange mode occurs under mild andenvironmentally friendly condition using an entirely aque-ous mobile phase. The elution from the monolith, whichmust contain ionizable ion-exchange functionalities, isachieved using a gradient of increasing salt concentrationin the mobile phase. The epoxide groups of a moldedpoly(glycidyl methacrylate-co-ethylene dimethacrylate)monolith readily react with many compounds to form ion

exchangers [129–131]. For example, the reaction withdiethylamine leads to an analog of the popular diethylami-noethyl (DEAE) chemistry, which is well suited even forlarge-scale separations of proteins. The breakthroughcurves measured for these monolithic columns with differ-ent proteins are very sharp and confirm the fast masstransport kinetics of the monoliths [132, 133]. The frontalanalysis used for the determination of the breakthroughprofile was also used for calculation of the dynamic capa-city of the column. For example, we found that the capa-city for the 60616 mm ID monolith at 1% breakthroughwas 324 mg of ovalbumin and represented the specificcapacity of 40.0 mg/g of separation medium or 21.6 mg/mL of column volume. Using commercial monolithic col-umns with a similar chemistry produced by Isco, Inc. forfast ion-exchange chromatography (Swift WAX), four pro-teins were easily separated in 3 min with an excellentresolution. These columns are also very stable and nochanges in separation and recovery have been foundeven after several hundreds of runs. This makes themwell suited for high throughput separation of biomacromo-lecules.

In addition to the monolithic DEAE weak anion exchanger,Isco, Inc. has also developed several other monolithic col-umns with chemistries including strong anion exchangeras well as weak and strong cation exchangers. It has beendemonstrated that, using these monolithic ion exchan-gers, resolution similar to that of conventional HPLC canbe readily achieved. Specific attention has also been paidto long-term stability of repeatedly used columns. Fig-ure 17 shows the separations of three proteins achievedover a long period of time and a large number of injec-tions.

High performance is in liquid chromatography oftensynonymous with high pressure since small size particlesare packed in the column. Column packed with largerbeads that run at medium pressure typically afford poorer


Figure 16. Rapid separation of ribonuclease A, cytochromec, myoglobin, and ovalbumin using monolithic poly(styrene-co-divinylbenzene) capillary column and a single step gradi-ent at 100 lL/min. Conditions: Column size 0.26100 mm.Mobile phase A, 0.1% TFA in 90/10 (v/v) water/acetonitrile;mobile phase B, 0.1% TFA in 10/90 (v/v) water/acetonitrile.Gradient profile that includes change from 100% mobilephase A to 100% B at time 0 is represented by the bolddashed line.

Figure 17. Test of stabilityof weak cation exchangemonolithic column SwiftWCX (Isco, Inc.). Condi-tions: column 5064.6 mmID, mobile phase gradientof 0.1 to 0.5 mol/L sodiumchloride in 0.01 mol/Lsodium phosphate bufferpH 7.6 in 4.5 min and to1 mol/L in 6.5 min, overallgradient time 11 min, flowrate 10 mL/min. Peaks:Ribonuclease (1), cytochro-me C (2), lysozyme (3).The two separations shownin this Figure wereachieved 503 runs apart.

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performance due to the mass transfer resistance withinthe “long” pores of these large diameter particles. In con-trast, monolithic media enable the high performance char-acteristic of HPLC columns packed with microparticles tobe achieved at a medium or even low pressure, thus offer-ing the separations in a unique mode that is called “highperformancemedium pressure liquid chromatography”.

In order to further accelerate the ion-exchange separa-tions, the rigid porous monoliths were provided with shortchains of poly(AMPS) grafted to the pore surface using acerium(IV) based initiating system (vide supra) [64]. In ourpreliminary experiments, the separations of myoglobin,chymotrypsinogen, and lysozyme in 10068 mm IDmono-lithic columns with these strong acid grafts were achievedin a linear gradient of the mobile phase within 2.5 min at aflow rate of 7 mL/min. Although this separation wasalready rather quick, removing the dead volume betweenthe peaks of chymotrypsinogen and lysozyme using non-linear gradients accelerated it even more and the proteinswere baseline separated within only 1.5 min.

A slightly different mechanism of proteins separationresults from the use of porous polymeric monoliths con-taining zwitterionic sulfobetaine groups [63, 69]. The ori-ginal approach involves photoinitiated copolymerizationof N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)-ammonium betaine and ethylene dimethacrylate. Alter-natively, the internal surface of porous poly(trimethylol-propane trimethacrylate) monoliths were grafted withzwitterionic “combs” by thermally initiated polymeriza-tion of the monomer within the pores. While the flowresistance of grafted monoliths was strongly affected bythe type of electrolyte, no changes were observed uponvariation in ionic strength of the mobile phase.

Another gentle method designed for the separation of pro-teins is hydrophobic interaction chromatography (HIC). Itsconcept is based on the interactions of surface hydropho-bic patches of proteins with hydrophobic ligands inter-spersed in the hydrophilic surface of the separation med-ium. The interaction occurs in an environment, such as anaqueous salt solution, that promotes these interactions.The column-bound ligands are typically short alkyl chainsor phenyl groups. The strength of the interaction dependson many factors, including the intrinsic hydrophobicity ofthe protein, the type of ligands, their density, the salt con-centration, and the temperature at which the separation iscarried out. In contrast to ion-exchange chromatography,the separation is achieved by decreasing the salt concen-tration in the mobile phase, causing the less hydrophobicmolecules to elute first.

Since the hydrophobicity of styrene- or alkyl methacrylate-based monolithic matrices is too high to make them usefulfor HIC, Xie et al. [65, 134] developed porous monolithsbased on highly hydrophilic copolymers of acrylamide and

methylenebisacrylamide. The hydrophobicity of the matrixrequired for the successful separations of proteins is con-trolled by the addition of butyl methacrylate to the polymer-ization mixture. The suitability of this rigid hydrophilicmonolith for the separation of protein mixtures wasdemonstrated on the rapid separation of five proteins inless than 3 min using a steeply decreasing concentrationgradient of ammonium sulfate.

Typically, proteins are eluted consecutively in HIC byapplying a decreasing gradient of salt concentration. How-ever in order to operate satisfactorily, a typical HIC columnmust be re-equilibrated in the initial mobile phase prior tothe next run. This decreases the number of runs that canbe performed within a given amount of time, and thusrepresents a serious limitation for high throughput pro-cesses. Therefore, we developed a new concept of hydro-phobic interaction chromatography which employs ther-mally induced change in surface polarity of the graftedcomposites to achieve the chromatographic separation ofproteins in a simple isocratic mode just by changing thetemperature of the column [74].

The preparation of monoliths with poly(NIPAAm) chainsgrafted to the internal pore surfacewas shownearlier in thispaper (Figure 7). The extended solvated chains that arepresent below the lower critical solution temperature(LCST) are hydrophilic, while the collapsed chains that pre-vail above theLCSTarehydrophobic. In contrast to isother-mal separations in which the surface polarity remains con-stant throughout the run, HIC separation of proteins can beachieved at constant salt concentrations (isocratic mode)while utilizing the hydrophobic-hydrophilic transition of thegrafted chains of poly(NIPAAm), which occurs in responseto changes in temperature. For example, carbonic anhy-drase and soybean trypsin inhibitor were easily separatedusing this column. First, the graftedmonolith was heated to408C, and a mixture of the two proteins was injected. Themore hydrophilic carbonic anhydrase was not retainedunder the experimental conditions, and eluted from the col-umn. In contrast, themore hydrophobic trypsin inhibitor didnot elute even after ten minutes. However, the elutionoccurred almost immediately once the temperature of thecolumnwas lowered to258C [74].

3.6 Separation of nucleic acids

To prepare media suitable for ion-exchange chromatogra-phy of nucleic acids,monolithic poly(glycidylmethacrylate-co-ethylene dimethacrylate) columns were modified todifferent extents by reaction with diethylamine to afford (1-N,N-diethylamino-2-hydroxy)propyl functionalities. Theirperformance was then demonstrated in the separations ofa homologous series of oligodeoxyadenylic (pd(A)12 – 18)andoligothymidylic acids (d(pT)12 – 24) at different flow rates.Very good separations of the oligonucleotides wereachieved, evenat thehigh flow rateof 4mL/min [135].



For the separation in reverse phase mode, monolithicpoly(styrene-co-divinylbenzene) columns were preparedinside a 200 lm ID fused silica capillary using a mixture oftetrahydrofuran and decanol as porogen. With gradientsof acetonitrile in 0.1 mol/L triethylammonium acetate,thesemonolithic columns allowed the rapid and highly effi-cient separation of single-stranded oligodeoxynucleotidesand double-stranded DNA fragments by ion-pairreversed-phase high-performance liquid chromatography(IP-RP-HPLC) [122, 136]. These authors also comparedthe performance of their monolithic columns with that ofmicropellicular, octadecylated poly(styrene-co-divinyl-benzene) beads and found a considerably better perform-ance for the former. The use of the monolithic columnenabled the analysis of an 18-mer oligodeoxynucleotidewith an efficiency of more than 190,000 plates/m. The ESIMS was on-line-coupled to the chromatographic separa-tion system to achieve detection of femtomolar amountsof 3 to 80-mer oligodeoxynucleotides. Similarly, double-stranded DNA fragments ranging in size from 51 to 587base pairs were also separated as demonstrated in Fig-ure 18. This method also allowed the sequencing of shortoligodeoxynucleotides.

The high resolving power of these monolithic capillary col-umns was also utilized for mutation screening in polymer-ase chain reaction amplified polymorphic loci. Recognition

of mutations was based on the separation of homo- andheteroduplexes by IP-RP-HPLC under partially denatur-ing conditions. This approach afforded characteristic peakpatterns both for homozygous and heterozygous sam-ples. Six different single nucleotide substitutions and com-binations thereof were confidently identified in 413 bpamplicons from six heterozygous individuals, each ofwhich yielded a different unique chromatographic profile[137]. The field concerning monolithic poly(styrene-co-divinylbenzene) capillary columns in the analysis of bothsingle- and double-stranded DNAnucleic acids by high-performance liquid chromatography-electrospray ioniza-tion mass spectrometry was reviewed recently [138].

4 ConclusionAlthough monolithic columns are a relatively new formatof stationary phases for HPLC and much remains to bedone, recent achievements have opened new vistas forthe preparation of an entirely new class of columns, alsocalled “stationary phases of the fourth generation”, withexactly tailored properties [139]. A short time ago, Guio-chon claimed [140]: “The invention and development ofmonolithic columns is a major technological change in col-umn technology, indeed the first original breakthrough tohave occurred in this area since Tswett invented chroma-tography, a century ago.” The large amount of experimen-tal work that has been done so far and the commercialavailability of various monolithic columns confirm thegreat potential of these new separation media. Theirunique properties, in particular the ease of their prepara-tion, their tolerance to high flow rates, and the rapid speedof chromatographic separations that can be achieved atacceptable back pressures, make the monolithic columnformat superior to the more common columns packed withbeads for some applications. Since monoliths are rather“young”, the number of different stationary phases, sep-aration mechanisms, and methods developed with thesemedia remains much smaller than that available forpacked columns. It is only a question of time until therange covered by the monolithic technology will beextended and successfully compete with all of the otherwell-established separation technologies. The extent ofthis review only allowed a description of the applicationsof monolithic columns in HPLC. However, these flow-through materials can also be seen to hold considerablepromise in other areas such as electrochromatography,microfluidics, gas chromatography, heterogeneous cata-lysis, and combinatorial chemistry [16].

Acknowledgment

Support of this work by a grant of the National Institute ofGeneral Medical Sciences, National Institutes of Health(GM-48364) is gratefully acknowledged.


Figure 18. High-resolution capillary ion-pair reversed-HPLCseparation of a mixture of double-stranded DNA fragmentsusing a 6060.20 mm ID monolithic poly(styrene-co-divinyl-benzene) capillary column (Reprinted with permission fromref. [136]. Copyright 2000 Elsevier). Mobile phase (A)100 mmol/L triethylammonium acetate, pH 7.0, (B) 20%acetonitrile in 100 mmol/L triethylammonium acetate, pH 7.0.Linear gradient 35–75% B in 3.0 min, 75–95% B in12.0 min, flow-rate, 2.2 lL/min, temperature 508C, UV detec-tion at 254 nm, sample pBR322 DNA-Hae III digest,1.81 fmol of each fragment.

764 Svec

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preparation and hplc applications of rigid macroporous organic polymer monoliths

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